The rifamycin derivatives AF/013 and AF/ABDMP and their side chain components, and derivatives AF/DNFI, and C-27 have been used against purified AMV reverse transcriptase. The derivatives inhibit the enzyme in a reversible manner. Complete inhibition is obtained when the derivatives are added to the enzyme before addition of template and primer. No inhibition is observed with the side chain components of AF/ABDMP and AF/013 even at high concentrations. When C-27 is added to the ongoing reaction of polymerization, the reaction proceeds uninhibited for at least one minute, even at high drug concentrations. Then, a progressive decrease of the rate of polymerization occurs, suggesting as previously reported for AF/ABDMP, that C-27 has no effect on chain elongation. On the contrary, AF/013 and AF/DNFI seem to act on chain elongation and initiation. Polyacrylamide gel electrophoresis of the DNA synthesized after addition of excess C-27 or AF/ABDMP to the polymerization reaction show no decrease in the size distribution of the product, confirming that the drugs act at the initiation step(s) of transcription. When the kinetic data of inhibition as a function of drug concentration are plotted according to Hill, a straight line was obtained in several experiments, using C-27. A biphasic plot was obtained with AF/DNFI, under identical experimental conditions, as already observed for AF/ABDMP.